JP6603662B2 - Protein thioether conjugation method - Google Patents

Description

  The present invention relates to an improved method for preparing protein conjugates with thioethers that use the free cysteine of the protein as the site of addition of the moiety of interest.

  Protein conjugates have diverse functionality, particularly in the pharmaceutical industry where the ability to covalently attach various moieties to the target therapeutic protein, thereby improving the properties of the molecule of interest, in particular.

  Peptide conjugation is usually obtained by solid phase synthesis, but this approach is less attractive for larger proteins. Where larger polypeptide / protein conjugation is desired, the process is complicated by the need to obtain site-specific conjugation to obtain a homogeneous product. Various approaches have been developed that provide a means for conjugation to N and C terminal residues as well as internal amino acid residues.

  With respect to growth hormone proteins, Gln and Lys residues have been successfully targeted as described in WO2005 / 070468 and WO2009 / 027369, etc., in which the specificity of the enzyme for specific internal amino acid residues Guarantees the site specificity of the conjugation.

  Site-specific conjugation of proteins containing growth hormone has been described previously, for example in WO99 / 03887, where site-specific conjugation is added by reacting the protein with a cysteine-reactive polymer or moiety. It is realized through a cysteine residue. Prior to this, the protein is partially reduced with dithiothreitol (DTT), taking care not to reduce to the disulfide bond of the protein, improving PEGylation with PEG maleimide (or PEG vinyl sulfone). The publication further states that it is difficult to obtain a high proportion of monoPEGylated protein in the final product composition.

  Various means for selectively reducing the added cysteine are also described in WO2006 / 134173, which deals primarily with factor Vlla, which also contains internal disulfide bonds. WO2010 / 089255 and WO2012 / 010516 disclose the use of additional cysteine residues for conjugation of albumin conjugates and cholic acid residues, respectively.

  Furthermore, cys conjugation can also be applied to proteins, antibodies or fragments thereof, as described in WO2007 / 03898 and WO2012 / 166622.

  The reduction and conjugation steps are chemical processes that are known per se, but are time consuming and inefficient because they require a lot of processing.

WO2005 / 070468 WO2009 / 027369 WO99 / 03887 WO2006 / 134173 WO2010 / 089255 WO2012 / 010516 WO2007 / 03898 WO2012 / 166622 WO2011 / 089250

  Thus, there is still a need to provide efficient, economical and applicable methods on an industrial scale for cys conjugate compounds to move from research to development.

In one aspect, the application is a method of preparing a protein conjugate in which a protein (P) is covalently bonded to a chemical moiety (Z) via a thioether.
a) obtaining a mixed disulfide composition comprising said protein;
b) adding a reducing agent to the protein composition;
c) causing reduction, and
d) obtaining a solution containing reduced protein (P-SH);
e) removing molecules from a solution having a molecular weight of less than 10 kDa;
f) adding an activated chemical moiety (Z *) to the solution containing the reduced protein;
g) causing a conjugation reaction;
h) obtaining a preparation of said conjugated protein (PSZ).

  The method may require the use of a cross flow filtration / tangential flow filtration system in certain embodiments. In particular, the reduction and conjugation reactions may be carried out in the retentate tank of the filtration system according to the present invention, and the filtration system can be used throughout the process if deemed preferred.

  The method may further comprise ultrafiltration and / or diafiltration steps. In situations where a defined concentration of one or more reactants is desired, an ultrafiltration step can be applied. As an example, the concentration of the mixed disulfide is adjusted by the ultrafiltration process to reach at least 100 μΜ, such as 150 μΜ, 250 μΜ, 350 μΜ, or even more than 400 μ 例 え ば, etc., regardless of the concentration of mixed disulfide in the starting preparation. can do. For growth hormone, the concentration may be at least 2 g / L, such as 5 g / L, or such as 10 g / L, regardless of the concentration of growth hormone in the starting preparation.

  Diafiltration can be used to remove small molecules from the filtration system, and reasonable thresholds such as 10 kDa can of course be used depending on the protein and reactant used.

  The method applies mixed disulfides as a starting point. This is because mixed disulfides are usually found to be a reliable source of proteins with free cysteine in the form of capped free cysteine, where Cap is derived from cysteine, cysteamine or glutathione. It is because it has been.

  The reducing agent and conditions for the reduction should be advantageous for the selective reduction of phosphines such as mixed disulfides and triarylphosphines, especially triphenylphosphine-3,3′-disulfonic acid disodium salt (TPPDS) is useful. Has been identified as a novel reducing agent. We have obtained a more efficient process when the concentration of reducing agent is at least equal to the concentration of protein (PSS-Cap), but the method of the present invention eliminates the need for high concentrations of reducing agent. I found.

  A halogenated moiety may be used as the activated chemical moiety (Z *). When an activated chemical moiety is added to the reduced protein (step f), at least one equivalent of activated chemical moiety (Z *) may be more advantageous compared to the amount of mixed disulfide. Found.

  As described herein, the method can be applied to the conjugation of a wide range of proteins and chemical moieties, providing an efficient and convenient way to obtain such protein conjugates. .

  The present invention can also solve additional problems that are apparent from the disclosure of the exemplary embodiments.

FIG. 6 shows the reduction of GH-L101C-S-glutathione at a concentration of 2.4 g / L with 5 equivalents of TPPDS, pH 7.4. The reaction is followed by AIE-HPLC, where the relative area% of starting material, reaction intermediate and product is plotted against the reaction time. ▲: GH-L101C-S-glutathione, X: reaction intermediate, ●: GH-L101C-SH. FIG. 1B is a diagram showing the conjugation of the reduced GH-L101C-SH with the side chain after reduction for 4 hours shown in FIG. 1A. 2.2 equivalents of side chains are added directly to the reduction mixture without any diafiltration. The relative area% of starting material and product is plotted against reaction time. The area% of the side chain is set to 100% at the start of the conjugation reaction. X: side chain, ●: GH-L101C-SH, ▲: GH-L101C-S- side chain, O: GH-L101C-S-glutathione, Δ: reaction intermediate. FIG. 5 shows the reduction of GH-L101C-S-glutathione to 5 g / L after ultrafiltration with 10 equivalents of TPPDS at pH 8.0 and 20 ° C. The reaction is followed by AIE-HPLC, where the relative area% of starting material, reaction intermediate and product is plotted against the reaction time. Δ: GH-L101C-S-glutathione, X: reaction intermediate, O: GH-L101C-SH. The figure represents the data from experiment 17 in Table 1. FIG. 3 is a diagram showing the conjugation between the side chain after reduction for 4 hours and reduced GH-L101C-SH shown in FIG. Three equivalents of side chains are added directly to the reduction mixture without any diafiltration. The relative area% of starting material and product is plotted against reaction time. The area% of the side chain is set to 100% at the start of the conjugation reaction. ж: Side chain, ●: GH-L101C-SH, ■: GH-L101C-S- side chain. FIG. 3 is a diagram showing the conjugation between the side chain after reduction for 4 hours and reduced GH-L101C-SH shown in FIG. After the reducing mixture is diafiltered into buffer 1, 3 equivalents of side chains are added to the reduced protein. The relative area% of starting material and product is plotted against reaction time. The area% of the side chain is set to 100% at the start of the conjugation reaction. ж: Side chain, O: GH-L101C-SH, □: GH-L101C-S- side chain. FIG. 6 shows the reduction of GH-L101C-S-glutathione to a concentration of 10 g / L after ultrafiltration with 5 equivalents of TPPDS, pH 7.4. The reaction is followed by AIE-HPLC, where the relative area% of starting material, reaction intermediate and product is plotted against the reaction time. Δ: GH-L101C-S-glutathione, X: reaction intermediate, O: GH-L101C-SH. FIG. 4B is a diagram showing the conjugation of the reduced side chain and reduced GH-L101C-SH for 4 hours shown in FIG. 4A. After the reducing mixture is diafiltered into buffer 3, 2.2 equivalents of side chains are added to the reduced protein. The relative area% of starting material and product is plotted against reaction time. The area% of the side chain is set to 100% at the start of the conjugation reaction. X: side chain, O: GH-L101C-SH, Δ: GH-L101C-S- side chain. FIG. 5 shows the correlation of minimum protein concentration during reduction as a function of ionic strength at three different temperatures. Experimental examples from Table 1 are represented by the following symbols: X: T = 5 ° C., Δ: T = 20 ° C. and O: T = 40 ° C. FIG. 3 shows the reduction of GH-L101C-S-glutathione under the conditions of Experiments 9, 12, 15 and 16 from Table 1. The reaction is monitored by AIE-HPLC and the relative area% of starting material is plotted against the reaction time. The solid line is 5 ° C and the dotted line is 40 ° C. FIG. 5 shows the correlation of minimum protein concentration during conjugation as a function of ionic strength at three different temperatures. Experimental examples from Table 2 are represented by the following symbols: X: T = 5 ° C., Δ: T = 20 ° C. and O: T = 40 ° C. It is a figure which shows the Example of the conjugation reaction by which the relative content of reduced protein (GH-L101C-SH) is monitored. FIG. 8A shows the relative content of reduced protein under the conditions of Experiments 5, 6 and 11 from Table 2. FIG. 8B shows the relative content of reduced protein during the conjugation reactions of experiments 14, 15, 18 and 19 from Table 2. The figure shows the conversion of reduced GH-L101C-SH (after diafiltration) and the addition of side chains. The reaction is monitored by AIE-HPLC and the relative area% of starting material is plotted against the reaction time.

SEQ ID NO: 1-human growth hormone AA1-181
FPTIPLSRLF DNAMLRAHRL HQLAFDTYQE FEEAYIPKEQ KYSFLQNPQT SLCFSESIPT PSNREETQQK SNLELLRISL LLIQSWLEPV QFLRSVFANS LVYGASDSNV YDLLKDLEEG IQTLMGRLED GSPRTGQIFK QTYSKFDTNS HNDDLRRKNY

Definitions The terms “polypeptide” and “peptide” as used herein mean a compound consisting of at least two amino acids linked by an amide (or peptide) bond.

  The term “amino acid” includes a group of amino acids encoded by the genetic code referred to herein as standard amino acids. Also included are natural amino acids that are not encoded by the genetic code, and synthetic amino acids.

  Commonly known natural amino acids include γ-carboxyglutamic acid, hydroxyproline, ornithine, sarcosine and phosphoserine. Commonly known synthetic amino acids are chemical synthesis of Aib (alpha-aminoisobutyric acid), Abu (alpha-aminobutyric acid), Tie (tert-butylglycine), β-alanine, 3-aminomethylbenzoic acid, anthranilic acid, etc. The amino acid produced by

  The term “protein” as used herein means a biochemical compound composed of one or more polypeptides.

  The term “growth hormone” is used to describe wild type growth hormone such as human growth hormone identified by SEQ ID NO: 1.

  The term “growth hormone variant” as used herein, a deletion, addition and / or substitution and / or addition of at least one amino acid residue that occurs in native human growth hormone. Means a growth hormone protein having an amino acid sequence derived from the structure of natural growth hormone, eg, the structure of human growth hormone identified by SEQ ID NO: 1. The term includes one or more amino acid residues in the growth hormone sequence replaced by other amino acid residues and / or one or more amino acid residues deleted from the growth hormone, and Also used for modified growth hormone proteins in which one or more amino acid residues are added and / or inserted into growth hormone.

  As used herein, the term “growth hormone compound” refers to a growth hormone molecule that retains at least some functionality of human growth hormone identified by SEQ ID NO: 1, as well as C53 to C165 in a growth hormone variant. , And this overall structure comprising two intramolecular disulfide bonds linking C182 to C189 or the corresponding amino acid residue. Such molecules may be growth hormone variants, growth hormone derivatives or growth hormone fusions, and growth hormone variant derivatives and fusions.

  As used herein, the term “growth hormone derivative” thus includes at least one added to one or more amino acids, such as one or more amino acid side chains of growth hormone or a growth hormone variant or analog. Human growth hormone or human growth hormone analog / variant containing two covalent modifications, in which the modifications are in the form of amides, carbohydrates, addition of alkyl groups, esters, PEGylation It is a body / variant.

  Such growth hormone derivatives may be referred to as “alkylated growth hormones”, including modification of growth hormones, optionally by addition of one or more chemical moieties to the growth hormone protein via a linker.

  The chemical moiety may be described as a property modifying entity. The chemical moiety may be fat soluble by including a fatty acid, and may optionally be linked to a growth hormone protein or analog via a linker. Chemical moieties that optionally contain optional linkers may be described as “side chains”. In this application where the chemical moiety is linked to a free cysteine, the “side chain” will be attached as an extension of the cysteine amino acid residue.

  The term “growth hormone fusion” as used herein expresses a growth hormone compound (wild type or variant) as a fusion protein with the polypeptide of interest and is therefore linked by traditional peptide bonds. Refers to protein molecules.

  The terms “drug”, “therapeutic agent”, “medicament” or “medicine” as used herein refer to an active ingredient used in a pharmaceutical composition that can be used therapeutically. .

  The term “ultrafiltration” is used to describe the process of filtering a solution using a suitable membrane or filter, and usually a pressure differential process results in a decrease in the volume of the solution, resulting in a solution in solution. The target molecule is concentrated.

  The term “diafiltration” is used to describe the process of filtering a solution using a suitable membrane or filter, where the solution discharged from the system is replaced with a new solution, so that the starting solution Is replaced with a new solution, the excipient of the solution is changed. In most systems, the solution is deemed to be replaced when five times the volume of the starting solution has been drained from the system, and the system is filled with an equal amount of fresh solution. This process moves the molecule of interest from one solution to a new solution.

  In one aspect, the invention relates to a method of preparing a protein conjugate in which protein (P) is covalently attached to chemical moiety (Z) via a thioether. The method may start with a composition of mixed disulfides containing proteins that are subjected to conjugation. This disulfide is reduced to obtain a protein with a free cysteine (P-SH) suitable for conjugation. The conjugation reaction is then performed by adding an activated chemical moiety (Z *) to the reduced protein to form a conjugated protein (P-S-Z). As described herein, the individual steps are optimized to obtain an efficient process for the preparation of protein conjugates.

  The present application relates to a method for preparing protein conjugates in which a protein (P) is linked to a chemical moiety (Z) via a sulfur atom that forms a thioether. As described herein below, the protein may be any protein of interest, including particularly therapeutic proteins such as growth hormone and growth hormone variants.

  In one embodiment, the present invention relates to a method of preparing a protein conjugate in which a chemical moiety is linked to a protein via a cysteine residue of the protein. The sulfur atom thus becomes the addition site for a chemical moiety that is added to the protein, whereby the protein and chemical moiety are covalently bonded via a thioether.

  “Free cysteine” has been found to be a suitable addition site for conjugation of various property-modifying groups. Free cysteine as used herein refers to a cysteine residue that does not participate in the normal disulfide bond between the two cysteines of one or two polypeptides. Usually, the free cysteine will be a cysteine that has not been introduced into the polypeptide sequence of interest by site-directed mutagenesis, but some proteins can alternatively contain a cysteine at the appropriate position. As described in the background art, the added cysteine can be a suitable addition site for a property modifying group to a protein. By introducing a cysteine residue, free cysteine is usually obtained because there is no partner in the protein to form a disulfide bond.

In one embodiment, the present invention provides a method of preparing a protein conjugate in which a protein (P) is covalently attached to a chemical moiety (Z) via a thioether.
a) obtaining a mixed disulfide composition comprising said protein;
b) adding a reducing agent to the composition to obtain a reduced mixture;
c) causing reduction, and
d) obtaining a solution containing reduced protein (P-SH);
e) optionally removing molecules of a solution having a molecular weight of less than 10 kDa;
f) adding an activated chemical moiety (Z *) to a solution containing the reduced protein to obtain a conjugation mixture;
g) causing a conjugation reaction;
h) obtaining a preparation of said conjugated protein (PSZ).

  The method allows for selective reduction of free cysteines, thereby allowing selective chemical conjugation of proteins.

Mixed disulfide disulfide (R1-SS-R2) is a covalent bond of two sulfur atoms that may be present in different (or the same) molecules. In proteins, cysteine residues can be linked through disulfide bonds, also called cystine.

  To be an effective target for the conjugation reaction, the free cysteine must be reduced. Proteins with free cysteines can be difficult to produce for the same reasons and are therefore often obtained as mixed disulfides containing small organic moieties. Mixed disulfides are molecules that contain disulfides that are similar to disulfide bonds between two cysteine amino acid residues, each contained in a polypeptide sequence (which may be the same or different). The small organic moiety is referred to herein as Cap and the mixed disulfide is thus a protein-S-S-Cap molecule. In this application, the term “mixed disulfide” is used for molecules that contain disulfide bonds that link two different entities, not both polypeptides, but the molecule is “normal” in addition to mixed disulfides. It may further contain a disulfide bond.

  In one embodiment, the method of the invention comprises a protein-S-S-Cap molecule reduction step since the protein subjected to conjugation is obtained in the form of a protein-S-S-Cap molecule composition.

  As described above, Cap is usually derived from a small organic moiety containing at least one sulfur atom that is part of the disulfide bond of the mixed disulfide. Such an organic moiety may be present as a monomer in the reduced form or as a dimer in the oxidized form. In mixed disulfides, -S-Cap is therefore the monomeric or half-dimer oxidized form. In one embodiment, -S-Cap is derived from cysteine / cystine, cysteamine / cystamine (which is decarboxylated cystine), or glutathione (G-SH) / glutathione disulfide (GS-SG) and is therefore a mixed disulfide In one embodiment, from protein-SS-cys, protein-SS-cyst, or protein-SSG (cys refers to half of cystine, cyst refers to half of cystamine, and G refers to half of glutathione disulfide). Selected. In other words, in one embodiment, the Cap of the protein-S-S-Cap is derived from cysteine, cysteamine or glutathione.

  As described above, the purpose of the reduction is to obtain a molecule having a free reduced cysteine (—SH) that is reactive in the conjugation reaction.

  In one embodiment, the mixed disulfide is a protein-S-S-Cap molecule derived from a protein where protein-S contains a free cysteine.

In one embodiment, the present invention provides a method of preparing a protein conjugate in which a protein (P) is covalently attached to a chemical moiety (Z) via a thioether.
a) obtaining a mixed disulfide composition comprising said protein;
b) adding a reducing agent to the protein composition;
c) causing reduction, and
d) obtaining a solution containing reduced protein (P-SH);
e) optionally removing molecules of a solution having a molecular weight of less than 10 kDa;
f) adding an activated chemical moiety (Z *) to the solution containing the reduced protein;
g) causing a conjugation reaction;
h) obtaining a preparation of said conjugated protein (PSZ).

  Each step is further described herein below and illustrated in the examples.

Growth Hormone Protein As described above, the purpose of reduction is to obtain a protein molecule having a free reduced cysteine (-SH) that is reactive in the conjugation reaction.

  In one embodiment, the mixed disulfide is a protein-S-S-Cap molecule derived from a protein where protein-S contains a free cysteine. In a further embodiment, the protein is growth hormone.

  The structure of the growth hormone protein consists of four helices (helix 1-4) linked to three loops (L1-3) and a C-terminal segment. In human growth hormone (SEQ ID NO: 1), helix 1 is defined by AA residues 6-35, helix 2 is defined by AA residues 71-98, helix 3 is defined by AA residues 107-127, and helix 4 is defined as AA residues 155-184.

  Since wild type human growth hormone does not contain free cysteines, the present invention is primarily directed to growth hormone variants containing additional cysteines that provide free cysteines. The method can be applied in the process of preparing growth hormone conjugates or derivatives using such free cysteines. Such derivatives can be obtained by alkylation of free thiol groups introduced through a single amino acid substitution of the growth hormone sequence.

  The growth hormone may in one embodiment be a growth hormone fusion, for example a protein molecule comprising a growth hormone sequence linked to a second protein sequence using a peptide bond. Fusions are usually obtained by expression of the fusion protein using a recombinant expression vector that links the DNA sequence encoding the growth hormone sequence with a DNA sequence encoding the second protein optionally including a linker sequence. Growth hormone fusions include, but are not limited to, fusions comprising antibody Fc regions and / or albumin proteins.

  The term “growth hormone compound” as used herein collectively refers to a growth hormone molecule that substantially retains the functional properties of mature human growth hormone identified by SEQ ID NO: 1. The compound may thus be a growth hormone derivative, including growth hormone, growth hormone fusion protein, growth hormone variant or analog, or acylated or alkylated growth hormone.

  In one embodiment, the growth hormone analog according to the invention comprises less than 8 modifications (substitutions, deletions, additions) compared to human growth hormone.

  In one embodiment, the growth hormone analog comprises less than 7 modifications (substitutions, deletions, additions) relative to human growth hormone. In one embodiment, the growth hormone analog comprises less than 6 modifications (substitutions, deletions, additions) compared to human growth hormone.

  In one embodiment, the growth hormone analog comprises less than 5 modifications (substitutions, deletions, additions) compared to human growth hormone. In one embodiment, the growth hormone analog comprises less than 4 modifications (substitutions, deletions, additions) compared to human growth hormone. In one embodiment, the growth hormone analog comprises less than 3 modifications (substitutions, deletions, additions) relative to human growth hormone. In one embodiment, the growth hormone analog comprises less than 2 modifications (substitutions, deletions, additions) compared to human growth hormone.

  In a series of embodiments, the growth hormone analog of growth hormone is at least 95, 96, 97, 98 or 99% identical to the human growth hormone specified by SEQ ID NO: 1.

Growth hormone compounds preferably have an increased plasma half-life (T _1/2 ) compared to wild type human growth hormone. This can be accomplished by various means known to those skilled in the art, such as amino acid substitutions that stabilize the protein from degradation. Increased circulation time of growth hormone compounds can also be obtained by covalent or non-covalent association with serum proteins. Serum albumin may be used by direct conjugation (optionally including a linker) or by protein fusion with growth hormone or this variant. Alternatively, chemical binding to albumin may also be examined in the same manner as fusion or linking with the antibody Fc region. Non-covalent binding to albumin may be obtained by use of an albumin conjugate such as growth hormone or an alkyl group covalently linked to this variant.

  In one embodiment, the growth hormone is a variant that is stabilized against proteolysis (due to specific mutations), and such variants are further alkylated at one or more amino acids of the growth hormone protein. Also good.

  Non-limiting examples of growth hormone proteins stabilized against proteolysis (by specific mutation) can be found in WO 2011/089250.

  Protease stabilized growth hormone protein variants include variants with additional disulfide bridges introduced. Additional disulfide bridges preferably link L3 to helix 2. This disulfide bridge can be obtained by introducing two extra cysteine amino acid residues. In a preferred embodiment, these cysteine amino acid residues replace the wild type amino acid residues at positions corresponding to AA84 or AA85 in H2 of SEQ ID NO: 1 and AA143 or AA144 in L3. Growth hormone variants are therefore according to the invention according to L73C / S132C, L73C / F139C, R77C / I138C, R77C / F139C, L81C / Q141C, L81C / Y143C, Q84C / Y143C, Q84C / S144C, S85C / Y143C, S85C in SEQ ID NO: 1. A pair of mutations corresponding to / S144C, P89C / F146C, F92C / F146C or F92C / T148C may preferably be included. In a further embodiment, the growth hormone variant comprises a pair of mutations corresponding to L81C / Y143C, Q84C / Y143C, S85C / Y143C, S85C / S144C or F92C / T148C in SEQ ID NO: 1.

  In one embodiment, the growth hormone is optionally monosubstituted / site specific, such as alkylation of one chemical moiety to the free cysteine introduced by the mutation, in addition to any protease stabilizing mutations described above. Is a growth hormone variant suitable for genetic modification. A non-limiting list of growth hormone variants suitable for alkylation can be found in WO2011 / 089255.

  In a further embodiment, the protein is a growth hormone variant that contains a free cysteine. In a further embodiment, the protein is a growth hormone variant comprising a free cysteine introduced into human growth hormone identified by SEQ ID NO: 1. In a further embodiment, the protein is T3C, P5C, S7C, D11C, H18C, Q29C, E30C, E33C, A34C, Y35C, K38C, E39C, Y42C, S43C, D47C, P48C, S55C, S57C, P59C, S62, E65C. , Q69C, E88C, Q91C, S95C, A98C, N99C, S100C, L101C, V102C, Y103C, D107C, S108C, D112C, Q122C, G126C, E129C, D130C, G131C, P133C, T135C, G136C, T142C, D147C, N149C A growth hormone variant comprising a cysteine mutation selected from the group of A155C, L156C, R178C, E186C, G187C and G190C. In a further embodiment, the protein is T3C, P5C, S7C, D11C, H18C, Q29C, E30C, E33C, A34C, Y35C, E88C, Q91C, S95C, A98C, N99C, S100C, L101C, V102C, Y103C, D107C, S108C. A growth hormone variant comprising a cysteine mutation selected from the group of D112C, Q122C and G126C.

  In further embodiments, the free cys mutation is located within AA 93-106 in hGH or the corresponding residue in the hGH variant. In further specific embodiments, the free cys mutation is located in L2, such as in AA99-106 or AA99-103 or in the corresponding residue.

  In a further embodiment, the free cys mutation is selected from the group of E30C, Y42C, S55C, S57C, S62C, Q69C, S95C, A98C, N99C, L101C, V102C and S108C.

  In a further embodiment, the single cys mutation is E30C. In a further embodiment, the single cys mutation is Y42C. In a further embodiment, the single cys mutation is S55C. In a further embodiment, the single cys mutation is S57C. In a further embodiment, the single cys mutation is S62C. In a further embodiment, the free cys mutation is Q69C. In a further embodiment, the free cys mutation is S95C. In a further embodiment, the free cys mutation is A98C. In a further embodiment, the free cys mutation is N99C. In a further embodiment, the free cys mutation is S100C. In a further embodiment, the free cys mutation is L101C. In a further embodiment, the free cys mutation is V102C. In a further embodiment, the free cys mutation is S108C.

  In a further embodiment, the protein is a growth hormone variant comprising a cysteine mutation selected from Y42C and L101C.

Reducing agent To obtain a protein containing reactive sulfur atoms, a reducing agent is added to the mixed disulfide composition to cause reduction, and the mixture is incubated to form a protein containing reactive sulfur atoms, eg, protein-SH. To obtain reduced protein. The described steps include: a) obtaining a mixed disulfide composition containing the protein, b) adding a reducing agent to the protein composition, c) causing reduction, and reducing protein (P-SH) It is the process of obtaining the solution containing.

  Only a few are mentioned herein because the reducing agent can be chosen between a plurality of available reducing agents and the skilled person knows that he can choose from a much larger repertoire of reducing agents. .

  In one embodiment, the reducing agent is a redox buffer selected from the group of glutathione, gamma-glutamylcysteine, cysteinyl glycine, cysteine, N-acetylcysteine, cysteamine and lipoamide.

  In one embodiment, a thiol disulfide redox catalyst such as an enzyme such as glutaredoxin is included.

  In one embodiment, the reducing agent is selected from small molecule reducing agents such as DTT.

  In one embodiment, the reducing agent is a substitution such as triphenylphosphine-3,3 ′, 3 ″ -trisodium trisulfonate (TPPTS) or the like or disodium triphenylphosphine-3,3′-disulfonate (TPPDS) or the like. A phosphine such as an aromatic phosphine such as a triarylphosphine such as a triarylphosphine.

  When the mixed disulfide is reduced, a solution containing reduced protein (P-SH) is obtained. It may be beneficial to remove the reducing agent and / or released Cap molecules prior to subsequent conjugation. In one embodiment, an optional step of removing small molecules, such as molecules having a molecular weight of less than 10 kDa, from a solution containing reduced protein (P-SH) may be included. In one embodiment, molecules having a molecular weight of less than 10 kDa are removed by diafiltration from a solution containing the reduced protein.

Chemical part (Z)
In the conjugation reaction, the chemical moiety is covalently bound to the sulfur atom of the free cysteine of the reduced protein (protein-SH).

  The chemical moiety may be any moiety suitable for conjugation with a protein, such as a property modifying moiety. A property modifying moiety may be a chemical moiety that can alter one or more characteristics of the protein of interest. In one embodiment, the chemical moiety is a property-modifying group such as a chemical moiety that can stabilize the protein and increase circulating half-life or increase potency. In one embodiment, the chemical moiety is an extender. In one embodiment, the chemical moiety (Z) is an albumin conjugate (AB). The chemical moiety may be used in an activated form (Z *) so that conjugation occurs effectively. In the method according to the invention as described hereinabove, an activated chemical moiety (Z *) is added to a solution containing the reduced protein, and conjugation of the chemical moiety with the reduced protein is: This results in the preparation of a conjugated protein (PSZ). Thus, the method according to the invention comprises the further steps: adding an activated chemical moiety (Z *) to a solution containing the reduced protein, causing a conjugation reaction, and said conjugate protein (PSZ) Obtaining a preparation of

In the summary of the above description, the invention provides a method of preparing a protein conjugate in which a protein (P) is covalently attached to a chemical moiety (Z) via a thioether,
a) obtaining a mixed disulfide composition comprising the protein;
b) adding a reducing agent to the protein composition;
c) causing reduction, and
d) obtaining a solution containing reduced protein (P-SH);
e) optionally removing molecules of a solution having a molecular weight of less than 10 kDa;
f) adding an activated chemical moiety (Z *) to the solution containing the reduced protein;
g) generating a conjugation reaction such as selective chemical conjugation;
h) obtaining a preparation of said conjugated protein (PSZ).

  The chemical moiety may be any moiety suitable for conjugation with a protein, such as a property modifying moiety. A property modifying moiety may be a chemical moiety that can alter one or more characteristics of the protein of interest. In one embodiment, the chemical moiety is a property-modifying group such as a chemical moiety that can stabilize the protein and increase circulating half-life or increase potency. In one embodiment, the chemical moiety is an albumin conjugate. The chemical moiety may be used in an activated form so that conjugation occurs effectively. In the method according to the invention as described hereinabove, the activated chemical moiety is combined with the reduced protein, and conjugating the chemical moiety to the reduced protein results in a conjugate protein via the sulfur atom. Is prepared.

  The chemical moiety is preferably an activated chemical moiety that means a moiety capable of reacting with protein-SH to form a protein-S-chemical moiety molecule. Such activated chemical moieties may include soft electrophilic alkylating reagents containing maleimide or haloacetyl groups known in the art.

  In one embodiment, the activated chemical moiety (Z *) is a halogenated chemical moiety (Z-halo) such as a halogenated property modifying group such as a halogenation extender. The halogenated chemical moiety (Z-halo) can include Br, I, or Cl. If desired, certain activation reactions may be performed prior to conjugation and prior to the addition of chemical moieties to the reduced protein.

  In one embodiment, the activated chemical moiety (Z *) is a halogenated albumin conjugate (AB-halo). In one embodiment, the chemical moiety (Z *) is a halogenated albumin conjugate comprising Br, I or CI as described in WO2010 / 089255. In one embodiment, the activated chemical moiety is iodoacetamide, such as iodoacetamide on the albumin conjugate side chain.

  In one embodiment, the activated chemical moiety (Z *) is a maleimide substituted chemical moiety (Z-maleimide) such as a maleimide substitution property modifying group such as a maleimide substitution extender. In one embodiment, the activated chemical moiety (Z *) is a maleimide substituted albumin conjugate as described in WO2010 / 089255.

  The reduction and conjugation reactions described in Examples 2 and 3 include the reaction of reducing growth hormone L101C with a halogenated albumin conjugate side chain where the halogen is iodine.

  In one embodiment, the albumin binding side chain is probably

Selected from.

  Further albumin binding side chains are described in WO2010 / 089255. In one embodiment, the activated albumin side chain is an iodide version of the above. Such iodide moieties can be obtained by dissolving a halogenated (Br or CI) chemical moiety in a KI (potassium iodide) solution to yield iodoacetamide.

  Further examples of chemical moieties include PEG molecules, serum albumin, albumin conjugates, Fc domains, growth hormone binding proteins, AA polymers [XTEN technology, Amunix and PASylation®, XL-protein], and heparosan And carbohydrate groups such as hydroxyethyl starch.

  As described herein below, the effectiveness of the conjugation step may depend on the ratio of reduced protein (P-SH) and activated chemical moiety (Z *).

Mixed Disulfide Composition Mixed disulfide compositions comprising proteins conjugated at free cysteine are preferably obtained as purified compositions containing only minor amounts of other proteins or impurities, and those skilled in the art will be able to It is believed that he knows how to produce and purify proteins with free cysteine as part.

  In one embodiment, the mixed disulfide composition has a protein concentration of 5-4000 μM, 25-1000 μM, 50-850 μM, 100-600 μM, 250-600 μM, or 250-500 μM. In further embodiments, the protein concentration is preferably greater than 100 μM, such as 150 μM, 250 μM, 350 μM, or even greater than 400 μM.

  In one embodiment, where the mixed disulfide is growth hormone or growth hormone variant, the concentration is preferably 0.1-100 g / L, 0.5-25 g / L, 1-20 g / L, 2-15 g / L, 5-15 g / L. Or 5 to 12 g / L or the like. In one embodiment, the hGH composition has a concentration of at least 2.5 g / L, such as at least 4.0 g / L, or such as at least 8.0 g / L, or at least 10 g / L. It is also noted that the concentration can be adjusted by including additional steps, whereby the mixed disulfide preparation is diluted or concentrated to reach the preferred concentration of protein. In one embodiment, the method includes a step of ultrafiltration of the mixed disulfide preparation to produce a composition of the mixed disulfide composition.

  In one embodiment, the mixed disulfide composition has a pH suitable for the reduction of the protein, such as a pH of 4-10, such as 5-9 or 6-8, and / or a pH suitable for the subsequently conjugated reduced protein. Have. In a further embodiment, the pH may be 7.0 to 8.0, or 7.2 to 7.8 or 7.3 to 7.6, such as approximately 7.4 to 7.5.

  In one embodiment, the mixed disulfide composition has a conductivity of about 5-50 mS / cm at 22 ° C., or such as 5-25 mS / cm at 22 ° C., or a conductivity of about 10 mS / cm at 22 ° C.

  In one embodiment, the mixed disulfide composition includes a buffer.

  In one embodiment, the mixed disulfide composition comprises a buffer selected from the group consisting of BES, HEPES, MES, phosphate, citrate, Bis-Tris and triethanolamine.

  In one embodiment, the mixed disulfide composition comprises 5-50 mmol / kg triethanolamine, such as 10-25 mmol / kg triethanolamine, or such as approximately 20 mmol / kg triethanolamine.

  In one embodiment, the mixed disulfide composition includes a salt.

  In one embodiment, the mixed disulfide composition comprises a salt selected from the group consisting of sodium salts, ammonium salts, guanidine salts, and potassium salts.

  In one embodiment, the mixed disulfide composition comprises a salt selected from the group consisting of sulfate, acetate, and halide salts.

  In one embodiment, the mixed disulfide composition comprises a salt selected from the group consisting of sodium sulfate, sodium acetate, ammonium acetate, guanidine hydrochloride, KI and NaCl.

  In one embodiment, the mixed disulfide composition comprises NaCl.

  In one embodiment, the mixed disulfide composition comprises 25-500 mmol / kg NaCl, such as 50-250 mmol / kg, such as 75-100 mmol / kg NaCl, or such as approximately 80 mmol / kg NaCl.

Method steps The components used in the method in the above summary are described with some variation. Additional items of variation within the scope of the present invention are the duration of the process steps, the use of various concentrations of each component, and the excipient of the solution in which the component is provided and the excipient of the solution in which each method step is performed It is.

  Those skilled in the art will appreciate that further modifications may be possible, but the following description further describes embodiments that the inventors have found particularly advantageous. Details illustrating the process can be found in the examples.

Addition of reducing agent Excess reducing agent in molar concentration is usually applied to have effective reduction of the mixed disulfide. Addition of a reducing agent to the mixed disulfide composition provides a reduced mixture. When the amount of reducing agent is 1 equivalent of the amount of mixed disulfide, the amount of reducing agent is the amount of mixed disulfide (or PSS-Cap) so that the molar concentration of mixed disulfide and reducing agent in the mixture is equal. It can be expressed in terms of equivalents.

  In one embodiment, the amount of reducing agent added in step b) is at least 2 equivalents of the amount of mixed disulfide (or PSS-Cap), such as 3-20 equivalents, such as 4-15 equivalents, or such as 5-10 equivalents and the like. In one embodiment, 2-12 equivalents of reducing agent is added. In one embodiment, 2 to 10 equivalents of reducing agent is added. In one embodiment, 3-7 or 4-6 equivalents of reducing agent is added.

  Since reducing agents can be a costly resource, it is advantageous to reduce the amount required, which is possible when the process steps are optimized as described herein. .

  An effective reduction reaction with a lower amount of reducing agent requires that the remaining reaction conditions be carefully selected, as provided by the present invention.

  In one embodiment, the amount of reducing agent is at most 10 equivalents of mixed disulfide, such as at most 8 equivalents, such as at most 5 equivalents, such as at most 4 equivalents of mixed disulfide to be reduced, such as at most 3 Equivalents, such as 2.5 equivalents at the maximum, such as 2 equivalents, such as 1.5 equivalents at the maximum.

  In one embodiment, the amount of reducing agent is 1-8 equivalents, such as 2-6 equivalents, of the mixed disulfide to be reduced.

  Reduction of the mixed disulfide may take several minutes or hours depending on the conditions. One skilled in the art will appreciate that different conditions provide different effectiveness. Thus, the time and conditions required to completely or nearly completely reduce the mixed disulfide are described in more detail herein below.

  The reducing agent may be added as a concentrate to the mixed disulfide composition, or simply by adding the reducing agent as a solid powder. The reducing agent is mixed with the mixed disulfide composition to initiate reduction. The mixture may be referred to as a reducing mixture.

  In order to have a sufficiently effective process, the reduction should result in a reduction of at least 80% of the total amount of mixed disulfide, such as a reduction of at least 90%. In such cases, reduction is considered sufficient if the amount of mixed disulfide is at most 20% of the amount of mixed disulfide in the reduction mixture, such as at most 10%. In a preferred embodiment, approximately 95% reduction of the mixed disulfide can be obtained leaving approximately 5% non-reduced mixed disulfide in the solution containing the reduced protein. In a further embodiment, an efficient process leaves up to 2% mixed disulfide within a reasonable amount of time.

  The reduction can occur over a period of at least 15 minutes, such as at least 30 minutes, or such as at least 1 hour. In one embodiment, the reducing mixture is left for 2 to 10 hours after addition of the reducing agent, such as 3 to 6 hours or approximately 3 to 4 hours.

  In one embodiment, the reduction is performed for up to 24 hours, such as up to 12 hours, such as up to 8 hours, such as up to 6 hours, such as up to 4 hours.

  In one embodiment, the reduction may be performed at 1 to 50 ° C., such as room temperature, such as 18 to 25 ° C. In an alternative embodiment, the reduction may be performed at a lower temperature, such as less than 10 ° C, such as approximately 4-6 ° C.

  Depending on the conditions applied, it may be advantageous to include a salt in the reduction mixture. The salt may be any salt or combination of salts such as those described with respect to the mixed disulfide composition.

  The inventors of the present invention provide a series of conditions that allow efficient reduction of mixed disulfides and describe the concentration of mixed disulfides used depending on temperature and salt (ionic strength of the reduced mixture). I used this information.

  The amount of salt in the solution can be described by ionic strength (I). This value is calculated from the concentration and charge of all ions present in the solution. For simplicity, protein and reducing agent charges are not included in the calculations according to the present invention, but buffer and salt components are included. As can be seen from the examples herein, an increase in ionic strength (mainly due to an increase in salt concentration) allows an efficient reaction under less favorable conditions (low temperature or lower concentration of reactants). .

As described in Example 5, the conditions for the reduction reaction are the correlation between the appropriate minimum protein concentration, the temperature, and the ionic strength with a maximum of 10 equivalents of reducing agent in the reduction mixture. Can be used to describe. The minimum protein concentration that allows efficient reduction is therefore the formula:
C _min = a * I ^-a1 exp (-b * T) where T is the temperature in degrees Celsius, I is the ionic strength of M (mol / L), and C _min is M (mol / L), the constants are a = 0.137 * 10 ⁻³ M ^1.425 , a ₁ = 0.425 and b = 0.070 ° C.− ¹ .

  Since many proteins are not very soluble at very high concentrations, the upper limit of mixed disulfide concentration can be a practical value. For most proteins, the reduction reaction is intended to function at concentrations up to 100 g / L.

  Since many salts are not very soluble at very high concentrations, the upper limit of the ionic strength of the reduction mixture can also be a practical value. For most salts, the reduction reaction is intended to function at concentrations up to 5M.

  Since many proteins are not soluble at very high temperatures, the upper temperature limit of the reduction mixture can be a practical value. For most proteins, the reduction reaction is intended to function at concentrations up to 50 ° C.

  According to this guidance, an effective reduction reaction can occur with up to 10 equivalents of reducing agent. It will also be apparent to those skilled in the art that individual proteins and reactions may require adjustments depending on their stability.

  In some embodiments, the concentration of the mixed disulfide in the reduction mixture is similar to the protein concentration of the mixed disulfide composition because the reducing agent is simply added to the composition in concentrated form. In one embodiment, the concentration of the mixed disulfide may be 5 to 4000 μΜ, 25 to 1000 μΜ, 50 to 850 μΜ, 100 to 600 μΜ, 250 to 600 μΜ, or 250 to 500 μΜ. In further embodiments, the concentration of the mixed disulfide is preferably greater than 100 μM, such as 150 μM, 250 μM, 350 μM, or even greater than 400 μM. In one embodiment where the mixed disulfide is growth hormone or growth hormone variant, the concentration is preferably 0.1-100 g / L, 0.5-25 g / L, 1-20 g / L, 2-15 g / L, 5-15 g / L, Or, for example, 5 to 12 g / L. In one embodiment, the concentration of GH is at least 2.5 g / L, or such as at least 4.0 g / L, or such as at least 8.0 g / L, or such as at least 10 g / L.

Intermediate Step Prior to proceeding with the conjugation step, the reduced protein (P-SH) can be converted into an excess reducing agent and / or a small organic molecule of mixed disulfide, e.g., a protein with a capped free cysteine (PSS-Cap) HS- It may be separated from a reducing mixture such as Cap. This optional step may be a step of removing molecules having a low molecular weight, such as molecules having a molecular weight of less than 10 kDa.

  Those skilled in the art will know various ways to remove small molecular weight compounds, such as by filtration through suitable membranes. In one embodiment, the method comprises a step of diafiltration using a membrane / filter having a 5 kDa, 10 kDa or 30 kDa cutoff.

  The cutoff can be selected by one skilled in the art depending on the size specificity required, knowing that a cutoff close to the molecular weight of the product increases the risk of losing the product. Roughly, it has a cut-off 1/3 to 1/6 of the molecular weight of the desired product.

  A further option before proceeding with the conjugation step is to change the solution of reduced protein (P-SH), for example to change the solvent, ie the individual excipient or excipient concentration of the solvent. In one embodiment, the method includes the step of changing the solvent of the solution of step d) prior to step f).

  A convenient way to change the solvent is diafiltration. In one embodiment, the diafiltration step is included to remove molecules in a solution having a molecular weight of less than 10 kDa and / or to change the solvent of a solution containing reduced protein (P-SH).

  The effectiveness of the diafiltration process, eg, the amount of small molecules and excipients removed, relates to the produced filtrate volume compared to the retentate volume. It is also noted that the word “remove” in this context should be interpreted as “reducing the concentration of”. This is because low molecular weight molecules and residual amounts of excipients are usually present after a diafiltration step (or alternative process step) that “removes” molecules with low molecular weight.

  The total volume of the composition when diafiltration is initiated is used as a reference to evaluate the required volume. Diafiltration is usually performed at a constant volume so that the volume of filtrate produced is equal to the volume of fresh solvent entering the system. In order for diafiltration to be effective, more than one volume of fresh solvent should preferably be used.

  In one embodiment, at least 2 volumes of solvent are applied to diafiltration, or at least 3, such as at least 4 or at least 5 volumes of solvent are applied to diafiltration.

  In one embodiment, the method according to the invention comprises the step of changing the solvent of the solution containing the reduced protein before initiating the conjugation reaction.

  In one embodiment, the method according to the invention comprises the step of changing the solvent of the solution of step d) before step f). In a further embodiment, the solvent is changed by diafiltration.

  In one embodiment, the reduced protein solution has a pH suitable for the protein, the resulting reduced protein, and the protein conjugate to be prepared, such as 4-10, such as 5-9 or 6-8. Changed to a solution or changed. In a further embodiment, the pH may be 7.0 to 8.0, or 7.2 to 7.8 or 7.3 to 7.6, such as approximately 7.4 to 7.5.

  In one embodiment, the reduced protein solution has a conductivity of approximately 1-150 mS / cm at 22 ° C., or such as 5-50 mS / cm such as 22 ° C., or such as 5-25 mS / cm such as 22 ° C., or 22 Changed or changed to a solution having a conductivity of approximately 10 mS / cm at 0C.

  In one embodiment, the reduced protein solution comprises or is changed to a solution comprising a buffer. In one embodiment, the buffer is selected from BES, HEPES, MES, phosphate, citrate, Bis-Tris and triethanolamine.

  In one embodiment, the solution of reduced protein comprises or contains a solution comprising 5-50 mmol / kg triethanolamine, such as 10-25 mmol / kg triethanolamine, or such as approximately 20 mmol / kg triethanolamine. Be changed.

  In one embodiment, the reduced protein solution comprises or is changed to a solution comprising a salt, such as any salt described with respect to the mixed disulfide composition.

  In one embodiment, the reduced protein solution comprises or is changed to a solution comprising a salt selected from the group consisting of sodium sulfate, sodium acetate, ammonium acetate, guanidine hydrochloride, KI and NaCl.

  In one embodiment, the reduced protein solution comprises or is changed to a solution containing NaCl.

  In one embodiment, the reduced protein solution comprises or is changed to a salt-free solution. In one embodiment, the reduced protein solution comprises or is changed to a solution without NaCl.

  In one embodiment, the reduced protein solution comprises or comprises a solution comprising 10-2000 mmol / kg NaCl, such as 50-500 mmol / kg NaCl, such as 75-100 mmol / kg NaCl, or such as approximately 80 mmol / kg NaCl. Change to solution.

Conjugation step As described above, conjugation is by a method performed by adding an activated chemical moiety (Z *) to a solution containing the reduced protein.

  If the previous reduction is not complete, the ratio of reduced protein to mixed disulfide (P-SH / P-S-S-Cap) may prevent high yielding conjugation reactions. In addition, the presence of excess reducing agent and released Cap molecules can interfere with the conjugation reaction.

  Again, the relative ratio of reactants, such as reduced protein and activated chemical moieties, affects the effectiveness of the reaction.

  In one embodiment, the molar concentration of the activated chemical moiety is at least equal to or perhaps twice the molar concentration of the conjugated protein. This can also be expressed in equivalents, such as at least 10 equivalents, such as 8 equivalents, such as 6 equivalents, such as 4 equivalents, such as 2 equivalents, or such as at least 1 equivalent, relative to the protein to be conjugated. Chemicalized chemical moieties (Z *) may be used. Since activated chemical moieties (Z *) can be a costly resource, it is advantageous to reduce the amount required, as described herein. This is possible if is optimized. An effective conjugation reaction using a reduced amount of activated chemical moiety (Z *) requires that the remaining reaction conditions be carefully selected, as provided by the present invention. In one embodiment, the amount of activated chemical moiety (Z *) is up to 8 equivalents of protein, such as up to 6 equivalents of conjugated protein, such as up to 4 equivalents, such as up to 3 equivalents, for example, 2.5 equivalents at the maximum, such as 2 equivalents, for example, 1.5 equivalents at the maximum.

  As described with respect to the reducing mixture, the solution of conjugation may be referred to as the conjugation mixture. With respect to the method steps a) to h) described above, a conjugation mixture is formed by the addition of the activated chemical moiety (Z *) in step f). It is further evident that the components of the conjugation mixture can be adjusted to optimize the conjugation conditions.

  Conjugation of reduced proteins with chemical moieties can take minutes or hours depending on conditions. One skilled in the art will appreciate that different conditions provide different effectiveness. Thus, the time required to obtain a complete or nearly complete conjugation will vary based on conditions as described in more detail herein below. The conjugation reaction according to this method is considered sufficient when the amount of starting material, for example reduced protein, reaches 10%, for example 5% or preferably 2% or less.

As described in Example 6, the conditions of the conjugation reaction include the appropriate minimum concentration of protein depending on temperature, ionic strength, and the activated chemical moiety (Z * used in the conjugation mixture). ) Can be described using the correlation between the relative quantities. The minimum protein concentration [C _{min of} M (mol / L) that allows efficient conjugation] is therefore C _min = a * exp (-b ₁ * Tb ₂ * I) + d * exp (-d ₁ * T), where T is the temperature in degrees Celsius, I is the ionic strength of M (mol / L), a = 6.96 * 10 ⁻⁴ M, b1 = 0.0396 ° C ⁻¹ , b2 = 10.9 M ⁻¹ , d = 6.12 * 10 ⁻⁵ M and d1 = 0.0289 ° C. ⁻¹ .

  As described above with respect to reduction, the upper limits of protein, ionic strength and temperature can be practical. For most proteins, the conjugation reaction is intended to function at concentrations up to 100 g / L. For most salts, the conjugation reaction is similarly intended to function at concentrations up to 5M. It is further contemplated that the conjugation reaction for most proteins will function at concentrations up to 50 ° C. Following this guidance, effective conjugation reactions can be obtained using up to 4 equivalents of activated chemical moieties (Z *).

  The activated chemical moiety may be added as a concentrate to the solution containing the reduced protein, or simply by adding the drug as a solid powder. In one embodiment, the activated chemical moiety is dissolved in a suitable solution prior to adding the activated chemical moiety to the solution containing the reduced protein. It is also possible that the chemical moiety is activated in solution prior to the conjugation reaction.

  The conjugation mixture according to the invention preferably has a pH of 5-10, such as 7.0-9.0 or 7.0-8.5.

  In one embodiment, the concentration of reduced protein in the conjugation mixture is at least 50 μM. In further embodiments, it is preferred that the concentration of the reduced protein in the conjugation mixture is greater than 100 μM, such as 150 μM, 250 μM, 350 μM, or even more than 400 μM.

  In one embodiment for conjugation of human growth hormone, the concentration is at least 1 g / L. In preferred embodiments, the concentration of human growth hormone is even higher, such as at least 2.0 g / L, such as at least 3.0 g / L, such as at least 5 g / L, or at least 7.5 g / L or at least 10 g / L. .

  According to the invention, the effectiveness of the conjugation can be improved by the salts contained in the conjugation mixture. This is particularly useful in lower protein concentration ranges, for example, when the concentration of reduced protein is 50 mM to 150 mM, or for human growth hormone, the protein concentration is 1-3 g / L in the conjugation mixture. It is of course possible to include a salt when higher protein concentrations are applied.

  The salt may be included in either the solution used in the conjugation mixture or a solution added separately to optimize reaction conditions. Salts are composed of cations and anions that provide equal positive and negative charges. Salts can be described as neutral, basic or acidic based on the ions obtained when hydrolyzed with water. The salt may be any salt, such as any salt described for mixed disulfide compositions. The salt is inorganic or organic such as sodium salt, ammonium salt, guanidine salt and potassium salt, sulfate salt, acetate salt and halide salt, or sodium sulfate, sodium acetate, ammonium acetate, guanidine hydrochloride, KI and NaCl. There may be.

  The amount of salt in the solution can be described by the ionic strength (I). This value is calculated from the concentration and charge of all ions present in the solution. For simplicity, any charges on proteins and activated chemical moieties (Z *) are not included in the calculations according to the present invention, but include buffer and salt components. As can be seen from the examples herein, the concentration of triethanolamine (buffer) and NaCl (or other salt) is included when calculating I.

  In one embodiment, the ionic strength (I) of the conjugation mixture is greater than 0.1M, such as greater than 0.2M, such as greater than 0.3M, such as greater than 0.4M, such as greater than 0.5M. As noted above, this is particularly useful when the concentration of reduced protein in the conjugation mixture is low, such as 50-250 μΜ, or such as 1-5 g / L or 0.5-3.0 g / L, for human growth hormone Can be.

  Similarly, the reaction conditions can be adjusted depending on the temperature of the conjugation reaction. In many situations, it may be advantageous to perform large scale reactions at or near room temperature (T = 18-25 ° C), but in other situations it may be preferable to raise or lower the temperature of the conjugation reaction for a variety of reasons. There is.

  In one embodiment, the conjugation reaction is performed at 15-50 ° C., such as in situations where the concentration of reduced protein is greater than 150 μM as described above. In an alternative embodiment, a lower temperature range may be applied if a salt is included in the conjugation mixture.

  In one embodiment where the conjugation reaction is performed at a temperature below room temperature, such as less than 20 ° C, such as less than 15 ° C, such as less than 10 ° C, or such as 2-8 ° C, the conjugation mixture is 0.1M It preferably has an ionic strength (I) of greater than, eg greater than 0.2M, such as greater than 0.3M, such as greater than 0.4M, such as greater than 0.5M.

  The activated chemical moiety is mixed with the reduced protein to initiate conjugation. The mixture is referred to herein as a conjugation mixture. Conjugation may occur for a period of at least 15 minutes, such as at least 1 hour, such as at least 2 hours, such as at least 3 hours, such as at least 4 hours, or such as at least 5 hours. In one embodiment, the conjugation mixture is left for 2 hours after addition of the activated chemical moiety, such as 3-6 hours or approximately 3-4 hours. In one embodiment, the conjugation mixture is allowed to stand for 2-20 hours, such as 6-16 hours or approximately 8-12 hours, etc. after addition of the activated chemical moiety.

  In one embodiment, conjugation may be performed for up to 24 hours, such as up to 18 hours, such as up to 12 hours, such as up to 6 hours, such as up to 4 hours.

  The conjugation reaction may be performed at various temperatures, such as 1-50 ° C, or such as 10-50 ° C. Conjugation may be performed at room temperature, such as 15-25 ° C. or 20-25 ° C., in one embodiment. In an alternative embodiment, the reduction may be performed at a lower temperature, such as less than 10 ° C, such as approximately 4-6 ° C.

  As the conjugation reaction proceeds, a preparation of conjugate protein (PSZ) is obtained, and once such a preparation is obtained, one skilled in the art will remove impurities with low molecular weight, as described above. One can proceed to further purification steps such as diafiltration of the product against the appropriate storage buffer, or more specific purification steps such as anion exchange chromatography.

Apparatus The apparatus used in the examples herein exemplifies a suitable apparatus for performing the method. This method can be carried out in a normal glass / tube or the like while being transferred as necessary. The present invention can also be very advantageously carried out in a cross flow filtration or tangential flow filtration (TFF) device. Such devices are well known in the art and allow the scale of the process to be extended from laboratory to industrial scale.

  In cross-flow filtration, the liquid is passed tangentially through the membrane / filter, the retentate and permeate or filtrate can be continuously collected / drained, and the retentate can be recycled to the system.

  As described in the examples, the system can be used for protein ultrafiltration and / or diafiltration at various stages in the conjugation process. Prior to the reduction, the disulfide preparation can be ultrafiltered to obtain a mixed disulfide composition at the desired concentration.

  Inlet and outlet pressures can be adjusted and new solutions containing buffer and / or other excipients can be added as the permeate is drained from the system.

  The system can also be used without applying a pressure differential. This means that the liquid volume is constant and the permeate is not drained from the system.

  One or more steps for the method of the invention are performed in one embodiment in an ultrafiltration / diafiltration system or a TFF system.

  In a further embodiment, the reduction (step c) is performed in a retentate tank of an ultrafiltration / diafiltration system or a crossflow filtration / TFF system.

  The reducing agent can be added to the retentate tank and circulation can be applied to mix the reducing agent with the mixed disulfide composition. During mixing, the excipients of the solution (including the mixed disulfide and reducing agent) are not changed and this volume remains constant. Mixing occurs in the system, but no filtration is applied (eg, no additional solution is put into the system) and / or no pressure differential is applied across the membrane, resulting in no permeate.

  In one embodiment, the permeate is not obtained or generated during the addition of the reducing agent and / or during the mixing of the reducing agent with the mixed disulfide. In one embodiment, the permeate is not obtained or produced during the reduction process. In one embodiment, diafiltration is not performed as part of steps b) and / or c).

  In a further embodiment, the conjugation (step g) is performed in a retentate tank of an ultrafiltration / diafiltration system or a crossflow filtration / TFF system.

  In one embodiment, a cross flow filtration / tangential flow filtration system is used throughout the process.

  In a further embodiment, the method comprises 1 for concentrating proteins, for removing molecules with a molecular weight of less than 10 kDa, or for changing the excipient or excipient concentration of the solution used. One or more ultrafiltration / diafiltration steps.

  When the method is finished, a preparation of conjugate protein (P-S-Z) (step h) can be obtained by emptying the cross-flow filtration system.

  The embodiments and examples described hereinbelow describe and illustrate certain features of the present invention. Those skilled in the art will now be able to conceive of many modifications, substitutions, alterations, and equivalents, and therefore these are also considered to be part of the present invention, so that the present invention is in any form. It should be understood that the invention is not limited to.

Embodiment
1. A method of preparing a protein conjugate in which a protein (P) is covalently bonded to a chemical moiety (Z) via a thioether,
a) obtaining a mixed disulfide composition comprising said protein;
b) adding a reducing agent to the protein composition to obtain a reduced mixture;
c) causing reduction, and
d) obtaining a solution containing reduced protein (P-SH);
e) optionally removing molecules of a solution having a molecular weight of less than 10 kDa;
f) adding an activated chemical moiety (Z *) to a solution containing the reduced protein to obtain a conjugation mixture;
g) causing a conjugation reaction;
h) obtaining a preparation of said conjugated protein (PSZ).

  2. The method of embodiment 1 performed in a cross flow filtration / tangential flow filtration system.

  3. The method according to embodiment 1 or 2, wherein the mixed disulfide is a protein having a capped free cysteine (P-S-S-Cap).

  4. The method of embodiment 3, wherein the composition of 4.a) has a concentration of P-S-S-Cap of at least 100 μΜ, such as 150 μΜ, 250 μΜ, 350 μΜ, etc., or even more than eg 400 μΜ.

  5. The method according to embodiment 3, wherein the composition of a) is obtained by ultrafiltration.

  6. The method of embodiment 3, wherein the Cap is derived from cysteine, cysteamine or glutathione.

  7. The method according to any one of embodiments 1 to 6, wherein the conjugation in step g) is a selective chemical conjugation.

  8. Substituted triarylphosphine such as triphenylphosphine-3,3 ', 3 "-trisulfonic acid trisodium salt (TPPTS) or triphenylphosphine-3,3'-disulfonic acid disodium salt (TPPDS) Embodiment 8. The method according to any one of Embodiments 1 to 7, wherein the phosphine is an aromatic phosphine such as a triarylphosphine.

  9. The method of any one of embodiments 1-8, wherein the amount of reducing agent is at least 2 equivalents of protein, such as 4-15 equivalents of protein, such as approximately 5-10 equivalents.

  10. The method according to any one of embodiments 1-9, wherein the reduction is performed in less than 10 hours.

  11. The method according to any one of embodiments 1-10, wherein the chemical moiety (Z) is a property modifying group such as an extender.

  12. The method of any one of embodiments 1-11, wherein the activated chemical moiety (Z *) is a halogenation extender.

  13. The method of any one of embodiments 1 to 12, wherein the activated chemical moiety (Z *) is a maleimide substitution extender.

  14. The method of any one of embodiments 1 to 13, wherein the chemical moiety (Z) is an albumin conjugate (AB).

  15. The method according to any one of embodiments 1-14, wherein the activated chemical moiety (Z *) is a halogenated albumin conjugate and the halogen is Br, I or Cl.

  16. The method according to any one of embodiments 1-15, wherein the activated chemical moiety (Z *) is a maleimide substituted albumin conjugate (AB).

  17. Embodiment 1 wherein an activated chemical moiety (Z *) is added in step f), such as up to 5 equivalents, such as up to 4, 3 equivalents or up to 2 equivalents compared to mixed disulfides. The method according to any one of 16 to 16.

  18. The method according to any one of embodiments 1-17, comprising the step of changing the solvent of the solution of step d) before step f).

  19. The method according to any one of embodiments 1-18, comprising step e).

  20. The method according to any one of embodiments 1-19, wherein step e) and / or solvent change step is performed by diafiltration.

  21. The method according to any one of embodiments 1-20, comprising the further step i) of removing molecules with a molecular weight of less than 10 kDa and / or changing the solvent of the preparation of step h).

  22. The method according to any one of embodiments 1-21, comprising a further step i) performed by diafiltration of the preparation of step h).

  23. The method according to any one of embodiments 1-22, wherein the one or more compositions of step a), the solution of step d) and / or the preparation of step h) comprises a buffer and a salt. .

  24. The method according to any one of embodiments 1 to 23, wherein the reduction mixture of step b) comprises a buffer and a salt.

  25. The method of any one of embodiments 1 to 24, wherein the conjugation mixture of the preparation of step f) comprises a buffer and a salt.

  26. The method according to any one of embodiments 1-25, wherein step i) removes the salt.

  27. The method according to any one of embodiments 1-26, wherein the buffer is triethanolamine.

  28. The method according to any one of embodiments 1-27, wherein the salt is NaCl and / or KI.

  29. The method according to any one of embodiments 1-28, wherein the composition of step a), the solution of step d) and the preparation of step h) have the same pH.

  30. The method according to any one of embodiments 1 to 29, wherein the composition of step a), the solution of step d) and the preparation of step h) have a pH of 7.0 to 8.0.

  31. The method according to any one of embodiments 1-30, wherein the solution of step d) and the preparation of step i) have different conductivities.

  32. The method according to any one of embodiments 1-31, wherein the conductivity of the solution of step d) is 10 mS / cm 2 (22 ° C.).

  33. The method according to any one of embodiments 1-32, wherein the conductivity of the preparation of step i) is 1-2 mS / cm (22 ° C.).

  34. The method according to any one of embodiments 1-33, wherein step e) is performed using a cellulose membrane, such as a 10 kD Hydrosart® membrane.

  35. The method according to any one of embodiments 1-34, wherein step i) is performed using a cellulose membrane, such as a 10 kD Hydrosart® membrane.

  36. The method of any one of embodiments 1-35, wherein at least one step is performed in a cross flow filtration / tangential flow filtration system.

  37. The method according to any one of embodiments 1-36, wherein the reduction (step c) is performed in a retentate tank of the ultrafiltration / diafiltration device.

  38. The method according to any one of embodiments 1-37, wherein the conjugation (step g) is performed in the retentate tank of the ultrafiltration / diafiltration device.

  39. The method of any one of embodiments 1-38, wherein a crossflow filtration / tangential flow filtration system is used throughout the process.

  40. The method according to any one of embodiments 1-39, wherein the preparation h) or i) is obtained by emptying the crossflow filtration system.

  41. The method of any one of embodiments 1 to 40, wherein preparation h) or i) has a concentration of at least 5 g / L.

  42. The method according to any one of embodiments 1-41, wherein the conjugate protein (PSZ) of preparation h) or i) is purified by ion exchange chromatography (AIEC) using Q sepharose HP etc. .

  43. Any of embodiments 1-42 wherein the conjugate protein (PSZ) of preparation h) or i) is concentrated using ultrafiltration resulting in a concentration of at least 10 g / L, such as 20 g / L. The method according to one.

  44. The method of any one of embodiments 1-43, wherein the free cysteine is introduced by a point mutation.

  45. The method of any one of embodiments 1-44, wherein the protein is a growth hormone polypeptide.

  46. Embodiment 1 wherein the free cysteine of the growth hormone polypeptide results from a point mutation selected from the group consisting of E30C, Y42C, S55C, S57C, S62C, Q69C, S95C, A98C, N99C, L101C, V102C and S108C. 45. The method according to any one of 45 to 45.

  47. The method of any one of embodiments 1-46, wherein the growth hormone polypeptide comprises an L101C point mutation.

48. The reduction mixture of step b) has a mixed disulfide concentration of at least C _min , wherein C _min is
C _min = a * I ^-a1 exp (-b * T)
Defined by, T is temperature in degrees Celsius, I is an ionic strength of reduced mixture (M), are ^{a = 0.137 * 10 -3 M 1.425} , a 1 = 0.425 and b = 0.070 ° C. ^-1 48. A method according to any one of embodiments 1-47.

The conjugation mixture of 49.f) has a reduced protein concentration of at least C _min , where C _min is
C _min = a * exp (-b ₁ * Tb ₂ * I) + d * exp (-d ₁ * T)
Where T is the temperature in degrees Celsius, I is the ionic strength (M) of the conjugation mixture, a = 6.96 * 10 ⁻⁴ M, b1 = 0.0396 ° C.- ¹ , b2 = 10.9 M ⁻¹ 49. The method of any one of embodiments 1-48, wherein d = 6.12 * 10 ⁻⁵ M and d1 = 0.0289 ° C. ⁻¹ .

General Method for Preparing Growth Hormone Proteins Having Free Cysteine Proteins can be expressed recombinantly in E. coli as described in WO 2011/089255.

  Briefly, E. coli cells expressing GH-L101C are isolated by centrifugation. The cell pellet is resuspended in Tris buffer containing EDTA and polysorbate 20. Cells are disrupted by passing through a high pressure homogenizer. The resulting homogenate is mixed with urea solution, pH adjusted and left overnight. The protein is thereby solubilized and natural glutathione is coupled with free cysteine to form GH-L101C-S-glutathione. Prior to conjugation with the reduced / albumin side chain, the GH-L101C-S-glutathione precursor can be purified by anion exchange chromatography.

  Protein is captured in an anion exchange step using Q Sepharose XL as stationary phase and Tris buffer as mobile phase. Elution is performed using a linear sodium chloride gradient. After adjusting the ionic strength, the eluted protein is purified by hydrophobic interaction chromatography. Use Phenyl Sepharose FF as the stationary phase and Tris buffer as the mobile phase. Elution is performed by applying a step gradient with varying sodium chloride concentration.

  The MEAE tag is removed by enzymatic digestion of the precursor to obtain GH-L101C-S-glutathione, which is finally purified by anion exchange after dilution. Source 30Q is used as the stationary phase and triethanolamine as the mobile phase. Elution is performed using a linear sodium chloride gradient. The protein is now ready for the conjugation process.

General Method for Preparing Albumin Conjugate Side Chains Albumin conjugate side chains can be synthesized as described in WO2011 / 089255.

  Prior to conjugation of the albumin conjugate side chain with GH-L101C-SH, the side chain was dissolved in a solution of KI (5M), ascorbic acid (50 mM), and triethanolamine (150 mM), pH 7.5, Activate the side chain. The concentration of side chains in the KI solution is usually between 5 and 30 g / L.

(Example 1) Reduction and conjugation without using TFF system
As GH-L101C-S-glutathione, one obtained by anion exchange chromatography as described above is used. The concentration is 2.4 g / L, NaCl is 0.08M, and triethanolamine is 0.02M (pH 7.4). The ionic strength is 0.09M.

  Add TPPDS (5 eq., About 0.12 g / g GH-L101C-S-glutathione) to GH-L101C-S-glutathione and perform AIE-HPLC for 4 hours every 30 minutes following reduction (autosampler Used, see FIG. 1A). After 4 hours, a side chain solution (2.2 eq, ˜0.12 g / g GH-L101C-S-glutathione) is added to the reduction mixture and AIE-HPLC is performed overnight following the conjugation reaction (see FIG. 1B).

Example 2 GH-L101C-SH reduction and conjugation apparatus using TFF system for ultrafiltration and diafiltration:
Millipore Labscale TFF system
AKTAcrossflow TFF system
Sartocon Slice 200 Filter, cut-off 10 kD [Hydrosart®]
solution:

  The preparation of GH-L101C-S-glutathione is obtained as described in the method above. The GH preparation is applied to a TFF system (Millipore Lab scale TFF system) and ultrafiltered until a concentration of 5 g / L is obtained. Reducing agent (TPPDS) is added to excess GH (10 equivalents, about 0.24 g / g GH-L101C-S-glutathione), placed in a retentate tank and circulated until uniform (about 15 minutes). The reduction mixture is left for 4 hours (without circulation). The reaction is followed by AIE-HPLC. As shown in FIG. 2, the reduction occurs within 4 hours under the application conditions.

  To assess the effect of including a diafiltration step, a sample (1 ml) of reduced GH-L101C-SH is taken before and after such a step and a separate conjugation reaction is performed.

  Reduced protein is obtained using a 10 kD [Hydrosart®] membrane that removes low molecular weight (<10 kda) molecules, diafiltration buffer 1 [conditions: 350 mL, 0.4 bar (inflow), 0.3 bar (outflow), and Dialyfilter with a constant volume of 5 volumes using a flux of 17LMH. The OD280 of the reducing solution was 23.7 due to TPPDS. The removal of TPPDS was followed at OD280. The permeate initially showed a high absorbance at 280 nM and dropped to 0.058. Finally, the retentate had an OD280 equal to the OD280 before the addition of TPPDS, indicating that TPPDS was removed.

  Conjugation was achieved by adding 1 mL of reduced protein (obtained before diafiltration) by addition of 0.1 mL side chain solution corresponding to the addition of 3 equivalents (approximately 0.16 g per GH-L101C-S-glutathione). The alkylation process is monitored by AIE-HPLC (Figure 3A).

  At the same time, using 1 mL of concentrated retentate (reduced GH-L101C-SH after diafiltration), 0.1 mL side chain corresponding to the addition of 3 equivalents (about 0.16 g per g of GH-L101C-S-glutathione) Add the solution to start the conjugation reaction. The conjugation reaction is monitored by HPCL (Figure 3B).

  The remaining concentrated retentate (66 mL) is mixed with the side chain solution and circulated through the TFF system to ensure mixing. The mixture is left overnight without pumping.

  To further purify the product, the conjugation mixture (retention solution) is diafiltered at a constant volume of 5 volumes against diafiltration buffer 2 to remove molecules in solutions with a molecular weight of less than 10 kDa. The product for any subsequent chromatography step is prepared.

  AIE-HPLC of the final diafiltration conjugate GH-L101C-S-side chain shows almost complete conversion of GH-L101C-SH and successful reduction in the amount of side chain and KI compared to the conjugation mixture (Not shown).

Example 3 Reduction and conjugation device for GH-L101C-SH using TFF system for ultrafiltration and diafiltration with high GH concentration and low side chain concentration:
Millipore Labscale TFF system
AKTAcrossflow TFF system
Sartocon Slice 200 Filter, cut-off 10 kD [Hydrosart®]
solution:

  The GH preparation is applied to the TFF system (AKTAcrossflow) as described above and ultrafiltered until an GH-L101C-S-glutathione concentration of 10 g / L is obtained (inlet pressure 2 bar, outlet pressure 1 bar). The same amount of TPPDS (5 equivalents) as in Example 1 is added to the retentate tank and circulated until uniform (about 15 minutes). Following the reduction, AIE-HPLC is performed every 30 minutes for 4 hours (see Figure 4A using an autosampler).

  After 4 hours, the reducing mixture is diafiltered 5 times against diafiltration buffer 3 at a constant volume. Add side chain solution (2.2 eq, about 0.12 g / g GH-L101C-S-glutathione) and apply circulation until uniform (about 15 min). AIE-HPLC is performed following the conjugation reaction (using an autosampler, see FIG. 4B).

  After the conjugation reaction, the conjugation mixture is reduced against diafiltration buffer 4 to reduce the content of KI and the remaining side chains and to prepare the conjugate protein for any subsequent chromatography step. Diafiltration through double volume.

Example 4 Method for Purifying Conjugate Protein GH-L101C-S- Side Chain Product after conjugation by anion exchange chromatography using Q Sepharose HP as stationary phase and triethanolamine as mobile phase. Can be purified. Elution can be performed with a linear sodium chloride gradient. The product can be subjected to diafiltration into a suitable storage buffer that removes impurities smaller in size than the 10 kDa membrane cutoff. The UF / DF process also allows the preparation of a concentrated product.

Overview of Examples 1-4
The reduction and conjugation process performed using the TFF system has proven successful. Three examples of reduction of GH-L101C-S-glutathione using TPPDS are shown and the results are shown in FIGS. 1A, 2 and 4A. FIG. 1A shows reduction with 5 equivalents of TPPDS at a protein concentration of 2.4 g / L. The conversion of starting material is only about 60% after 4 hours. However, when the reduction is performed at 5 g / L with 10 equivalents of TPPDS (FIG. 2), or 10 g / L with 5 equivalents of TPPDS (FIG. 4A), the reaction is complete in less than 4 hours. It has also been observed that higher substrate (protein precursor) concentrations can reduce the amount of reducing agent.

  The above examples include some proof that the conjugation reaction can be performed in a TFF system (FIGS. 3A, 3B and 4B). In all examples, the reduction was carried out for 4 hours.

  The conjugation shown in FIG. 1B resulted in less than 60% conjugation after 20 hours. As shown in FIG. 1A, the reduction of the starting material is incomplete, so the remaining GH-L101C-S-glutathione and the reaction intermediate may interfere with the conjugation reaction. During the reaction period, GH-L101C-S-glutathione and the reaction intermediate were completely converted to GH-L101C-SH, but no complete conversion to GH-L101C-S-side chain occurred. The side chain is thought to be depleted by reaction with glutathione and possibly the remaining TPPDS before it can react with the free thiol of the protein.

  The only difference between FIGS. 3A and 3B is the intermediate diafiltration step, which increases the yield of conjugation to more than 90% compared to less than 80%.

  It can also be seen that intermediate diafiltration affects the side chain concentration. Without diafiltration, the side chain concentration drops almost immediately, but the performance of the intermediate diafiltration step avoids this sharp drop in side chain concentration.

  The decrease in side chain concentration may be due to side chain reaction with released glutathione (corresponding to one equivalent of side chain) and possibly TPPDS. With the introduction of intermediate diafiltration, these side reactions can no longer take place.

  Another advantage of the combination of ultrafiltration and diafiltration is illustrated by comparing FIGS. 3B and 4B. In FIG. 3B, the addition of 3 equivalents of side chains and the use of pH 8.0 has resulted in a fast almost complete conjugation reaction. A lower pH is advantageous because the protein is not very stable at pH 8.0. However, lower pH will also result in lower reaction rates. Similarly, the use of side chains should be minimized, but the reduction of side chains will also reduce the reaction rate. FIG. 4B shows that by increasing the protein concentration during the conjugation reaction, a fast enough reaction can be obtained with the use of fewer side chains and lower pH.

  Since conjugated proteins, such as those obtained as a conjugation mixture, are not very stable, it may be advantageous to include additional purification steps. As described in Example 4, the introduction of a final diafiltration step results in a stable protein preparation that can be stored under normal conditions, and further, the protein preparation can be obtained without dilution or pH adjustment. Thus, further processing can be avoided and applied directly to subsequent columns.

Example 5 Definition of Minimum Protein Concentration for Reduction Reaction The inventors of the present invention have found that an important parameter to be controlled is the concentration of protein in the reduction reaction mixture. As noted above, in situations where the starting material is at a relatively low concentration, the reaction can be improved by including a concentration step prior to adding the reducing agent. Concentration of the protein composition allowed for sufficient reduction using a smaller equivalent of reducing agent. Further tests have shown that the reaction rate is also affected by the temperature and ionic strength of the reduction mixture.

  A number of conditions were examined to describe the combined effects of protein concentration, temperature and ionic strength of the reducing mixture (Example in Table 1). As a reference, triethanolamine (20 mM) and NaCl (80 mM) were included with an ionic strength of 0.09. Experiments 4, 5, 10, 11 and 16 increased the ionic strength, including additional salt, while 14 and 15 decreased the salt concentration. The ionic strength of the buffer is calculated as a fraction of the charged buffer multiplied by the total concentration of the buffer. The fraction (x) of the charge buffer is the Henderson-Hasselbalch equation:

To calculate from the pKa of the buffer.

  Based on the reaction kinetics, a mechanism model representing reduction was developed. In addition to the inherent effects of protein concentration and reducing agent molar ratio, the model includes the effect of conductivity and temperature on the reaction rate constant. Model parameters were determined according to the experimental data. Model simulation was then used to represent the combined effects of protein concentration, temperature, and ionic strength in a number of conditions.

  A correlation was obtained representing the minimum concentration required to fully reduce at a given temperature and ionic strength. Sufficient in this case means that the concentration of the starting protein drops to <2% within about 6 hours. The large number of data points generated using simulations allowed for detailed correlations and hence unique (unusual) forms of correlation.

The following correlation is derived and illustrated by FIG.
C _min = a * I ^-a1 exp (-b * T), where
T is the temperature in degrees Celsius
I is the ionic strength of M (mol / L)
C _min is M (mol / L) and the constant is
a = 0.137 * 10 ^-3 M ^1.425
a ₁ = 0.425
b = 0.070 ° C ^-1 .

The calculation of C _min for Table 9 and experiments 9 and 16 from FIG. 9 is now illustrated below.
C _min is calculated by the following correlation described above, C _min = a * I ^−a1 exp (−b * T).
Calculation for Experiment 9
I = 0.08 (NaCl) + 0.02 * 1 / (1 + 10 ^(7.4〜7.8) ) (triethanolamine) = 0.09M
T = 5 ℃:
C _min = 0.137 * 10 ^-3 * 0.09 ^-0.425 * exp (-0.070 * 5) = 0.26 * 10 ^-3 M = 0.26mM
C _P of the experiment 9 was 0.11mM. This is below C _min and is therefore outside the range of preferred conditions according to the present invention.
Calculation for Experiment 16
I = 0.516 (NaCl) + 0.004 * 1 / (1 + 10 ( ^7.4-7.8 )) (triethanolamine) = 0.52M
T = 40 ℃:
C _min = 0.137 * 10 ^-3 * 0.52 ^-0.425 * exp (-0.070 * 40) = 0.01 * 10 ^-3 M = 0.01mM
In C _P 0.02 experiment 16, which exceeds the C _min, it is in the range of preferred conditions according to the present invention.

The utility is further evident from FIG. 6 which shows two experiments where the protein concentration (C _P ) is higher than C _min (experiments 12 and 16) and two experiments where C _P is lower than C _min (experiments 9 and 15) is there. In experiments 12 and 16, the starting material (protein-SS-Cap) is converted to less than 2% within 6 hours reaction time. Experiments 12 and 16 have useful reaction yields, either by high protein concentrations to compensate for low temperatures (Experiment 12) or by having both high ionic strength and higher temperatures to compensate for low protein concentrations (Experiment 16). Is obtained.

Example 6 Definition of Minimum Protein Concentration for Conjugation Reaction As shown in the previous examples (e.g. Example 3), the use of higher protein concentrations in the conjugation reaction is sufficient A conjugation reaction was obtained, which allowed the use of a smaller equivalent of activated chemical moiety (Z *), as exemplified by the albumin binding side chain. Again, the effects of ionic strength and temperature were examined. A sufficient conjugation reaction in this case means that the concentration of the starting protein (protein-SH) falls to <5% within about 10 hours. As a standard, triethanolamine (20 mM), NaCl (80 mM) and KI (0.5 M) were included under the condition of an ionic strength of approximately 0.5.

The conjugation reaction conditions include protein concentration (C _p ), temperature in degrees Celsius (Temp), number of side chain equivalents (n), and ions in the reduction mixture (including buffer but excluding protein and side chain components). Strength, such as salt concentration, is included. Based on the conjugation conditions tested in Experiments 1-20 in Table 2, a mechanistic model was developed using the same approach as for reduction. Use simulation to generate a significant amount of data, thereby obtaining a correlation that represents the minimum protein concentration required to obtain sufficient conjugation at any combination of a given temperature and ionic strength Became possible. Sufficient in this case means that the reduced protein concentration drops to <5% within about 10 hours. Based on the data obtained, suitable conditions for the conjugation reaction can be defined using the following definition for the minimum protein concentration (C _min ) depending on temperature and ionic strength illustrated by FIG.
C _min = 6.96 * 10 ^-4 * exp (-0.0396 * 5-10.9 * I) + 6.12 * 10 ^-5 * exp (-0.0289 * 5)
= 6.96 * 10 ^-4 * exp (-1.98-10.9 * I) + 5.30 * 10 ^-5
C _min = a * exp (-b ₁ * Tb ₂ * I) + d * exp (-d ₁ * T), where T is the temperature in degrees Celsius and I is M (mol / L) Ionic strength, C _min is the minimum protein concentration of M (mol / L)
a = 6.96 * 10 ^-4 M
b ₁ = 0.0396 ° C ^-1
b ₂ = 10.9M ^-1
d = 6.12 * 10 ^-5 M
d ₁ = 0.0289 ° C. ⁻¹ .

Calculations for conjugation experiments 15 and 18 are shown below:
Calculations for Example 15
I = 0.08 (NaCl) +0.75 (KI) + 0.02 * 1 / (1 + 10 ^(7.4-7.8) ) (triethanolamine) = 0.84M
T = 5 ℃:
C _min = 6.96 * 10 ^-4 * exp (-0.0396 * 5-10.9 * 0.84) + 6.12 * 10 ^-5 * exp (-0.0289 * 5) = 0.05 * 10 ^-3 M = 0.05mM
The protein concentration (C _P ) in experiment 15 was _{0.45 M} , clearly exceeding the calculated C _mim .

Calculation for Experiment 18
I = 0.027 (NaCl) +0.07 (KI) + 0.007 * 1 / (1 + 10 ^(7.4〜7.8) ) (triethanolamine) = 0.10M
T = 40 ℃:
C _min = 6.96 * 10 ^-4 * exp (-0.0396 * 40-10.9 * 0.10) + 6.12 * 10 ^-5 * exp (-0.0289 * 40) = 0.07 * 10 ^-3 M = 0.07mM
The protein concentration (C _P ) for experiment 18 was 0.04 mM, which is less than C _{min of} 0.10 M.

The usefulness of the correlation is evident from FIGS. 8A and 8B, which show a series of experiments that test various conjugation conditions. In experiment 11, the amount of reduced protein decreases to approximately 5% of the starting material after about 10 hours at 20 ° C. (C _P = 0.08 mM and 3.6 equivalents of side chain). In Experiment 5, the reaction obtained by increasing the amount of reduced protein (C _p ) is faster even if the number of equivalents of side chains is reduced to 2 equivalents. Compared to Experiment 5, as shown by Experiment 6, increasing the ionic strength further accelerates the reaction. In experiments 14, 15 and 19 (FIG. 8B), the protein concentration (C _P ) is higher than C _min . On the other hand, Experiment 18 shows that the effectiveness of the experiment with C _P less than C _min is low. In the latter, the reaction was very slow. After 20 hours, about 20% of starting material still remains. In experiment 19, the ionic strength increases, but C _P and T remain constant compared to experiment 18, resulting in a higher reaction rate and thus the starting material is converted to less than 5% in less than 3 hours. Is done. The two examples at 5 ° C both have a C _P greater than C _min and the starting material is converted to less than 5% in less than about 10 hours. However, an increase in _CP and ionic strength in Experiment 15 compared to Experiment 14 resulted in an even more efficient conversion of the starting material.

  While particular features of the invention have been illustrated and described herein, many modifications, substitutions, changes, and equivalents will now occur to those skilled in the art. Therefore, it is to be understood that the appended claims are intended to cover all such modifications and changes as fall within the true spirit of the invention.

Claims (13)

A method of preparing a protein conjugate in which a protein (P) is covalently attached to a chemical moiety (Z) via a thioether comprising:
a) a step of obtaining a mixed-disulfide compositions, a process wherein the mixed disulfide is a protein having a capped free cysteine (PSS-Cap), the concentration of the mixed disulfide is at least 2.5 g / L,
b) a pre-SL was added phosphine mixed-disulfide composition to obtain a reduction mixture, the amount of phosphine reduction mixture is a step which is up to 10 equivalents of the concentration of mixed disulfides,
c) causing a reduction reaction;
d) obtaining a solution containing reduced protein (P-SH);
e) removing molecules from a solution having a molecular weight of less than 10 kDa;
f) adding an activated chemical moiety (Z *) to a solution (including reduced protein) to obtain a conjugation mixture comprising 1 to 4 equivalents of the chemical moiety (Z *);
g) causing a conjugation reaction;
h) obtaining a preparation of said conjugated protein (PSZ),
Least as much as engineering a) and e) are carried out in a tangential flow filtration system, method. 2. The method of claim 1 , wherein Cap is derived from cysteine, cysteamine or glutathione. The reduction mixture of step b) has a mixed disulfide concentration of at least C _min , where C _min is
C _min = a * I ^-a1 exp (-b * T)
Defined by, T is temperature in degrees Celsius, I is an ionic strength of reduced mixture (M), are ^{a = 0.137 * 10 -3 M 1.425} , a 1 = 0.425 and b = 0.070 ° C. ^-1 The method according to claim 1 or 2 . The conjugation mixture of f) has a reduced protein concentration of at least C _min , where C _min is
C _min = a * exp (-b ₁ * Tb ₂ * I) + d * exp (-d ₁ * T)
Where T is the temperature in degrees Celsius, I is the ionic strength (M) of the conjugation mixture, a = 6.96 * 10 ⁻⁴ M, b1 = 0.0396 ° C.- ¹ , b2 = 10.9 M ⁻¹ is d = 6.12 * 10 ^-5 M and d1 = 0.0289 ℃ ^-1, method according to any one of claims 1 to 3. The method according to any one of claims 1 to 4 , wherein the phosphine is triarylphosphine. 6. The method of claim 5 , wherein the triarylphosphine is triphenylphosphine-3,3′-disulfonic acid disodium salt (TPPDS). 7. The method according to any one of claims 1 to 6 , wherein the activated chemical moiety (Z *) is a halogenated albumin conjugate comprising Br, I or Cl. 8. The method according to any one of claims 1 to 7 , wherein the conjugation mixture of step f) comprises up to 3 equivalents of activated chemical moiety (Z *) compared to the mixed disulfide. 9. The method according to any one of claims 1 to 8 , wherein step e) is diafiltration performed using a cellulose membrane. 10. The method of claim 9 , wherein the diafiltration buffer does not contain a reducing agent. Reduced mixture of mixed disulfides, step b) step a), step d) the solution, the conjugation mixture in step f), and / or preparation of step h) is triethanolamine, of claims 1 to 10 The method according to any one of the above. The mixed disulfide of step a), the reducing mixture of step b), the solution of step d), the conjugation mixture of step f), and / or the preparation of step h) has a pH of 7.0 to 8.0. the method according to any one of 11. 13. The method according to any one of claims 1 to 12 , wherein the protein is a growth hormone polypeptide comprising an L101C point mutation.

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